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9-Oxononanoyl cholesterol, a cholesterol core-aldehyde formed during lipoprotein oxidation, was recently identified in advanced human atherosclerotic lesions. Here we present a rapid and sensitive HPLC method for 9-oxononanoyl cholesterol analysis. 9-Oxononanoyl cholesterol was converted to the corresponding fluorescent decahydroacridine derivative by reaction with 1,3-cyclohexanedione. The derivatives formed were purified by solid-phase extraction on C-18 columns, separated by reversed phase HPLC with isocratic elution, and detected by their fluorescence. Decahydroacridine derivatives of 9-oxononanoyl cholesterol were stable for at least 160 h. The limit of quantitation of the method presented is at the low ( Ϸ 50) femtomole level, with an absolute limit of detection (signal: noise ‫؍‬ 6) of 15 fmol. Intra-assay variation was Յ 5%, while interassay variations were between 5 and 15%, depending on the concentration of the analyte. Standard curves were linear over nearly three orders of magnitude (50 fmol-12.5 pmol). 9-Oxononanoyl cholesterol proved to be the major cholesterol core-aldehyde formed during t-BuOOH/FeSO 4 oxidation of cholesteryl linoleate and Cu 2 ϩ -induced LDL oxidation, findings confirmed by atmospheric pressure chemical ionization-mass spectrometry. Analysis of lipid extracts obtained from advanced human atherosclerotic lesions revealed the presence of 9-oxononanoyl cholesterol in all tissue samples analyzed (28 ؎ 14 mol/mol cholesterol, n ‫؍‬ 9) despite the presence of ␣ -tocopherol (4 ؎ 1.2 mmol/mol cholesterol, n ‫؍‬ 9).

A new route for the preparation of 9-oxononanoyl cholesterol (5) and its stable dimethylacetal (4) is described. The core aldehyde 5 is one of the major products formed during lipid peroxidation. The synthesis starts with the ozonization of oleic acid in methanol and further reduction with dimethyl sulfide to yield 9,9-dimethoxy nonanoic acid (2a). The condensation of 2a with cholester ol is achieved with N,N -dicyclohexylcarbodiimide in dichloromethane to give 4. Further hydrolysis of 4 with the help of an acidic ion exchange resin yields 9-oxononanoyl cholesterol.-Boechzelt, H.

In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called ‘ERROR-FFPE-DNA’ that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.